Ebola virus is one of the most lethal infectious threats to mankind. These infections cause severe hemorrhagic fevers in humans and non-human primates and produce mortality rates of up to 90%. Ebola virus is classified in the NIAID Biodefense Research Agenda for CDC as a Category A Agent (USDHHS-NIH) and is a biosafety level 4 (BSL4) pathogen. There are four species of the Ebola virus, and there is currently no vaccine or antiviral therapy against Ebola virus infections for humans. The elusive nature of the natural reservoir makes eradication of the agent impossible and elevates the control of this virus and bioterrorist threat through vaccination or therapeutic means to a level of paramount importance. Currently, only heroic, symptomatic measures are available for the treatment of Ebola infections. The overall goal of this research is to address this critical need by developing small molecule, orally active antidotes to Ebola virus infections, which can be used prophylactically or in the 4-16 day post-infection period when fever and hemorrhagic symptoms of disease are present. The cysteine proteinase cathepsin L, a lysosomal enzyme, has been reported recently to be a new target for inhibition of SARS virus infections. Furthermore, inhibitors of cathepsin L can prevent SARS coronavirus entry into target cells. Ebola virus and SARS virus both employ cathepsin L activity for viral entry and cathepsin L is strongly implicated in the processing of the Ebola virus glycoprotein. Our strategy is to take advantage of this discovery to develop inhibitors of this new anti-Ebola target, cathepsin L, which is required for virus entry. This innovative approach involves targeting a host enzyme rather than a viral component. In preliminary studies, we built a pseudovirus carrying the Ebola glycoprotein, which can be studied under BSL2 containment, and demonstrated the capability of the pseudovirus to infect 293T cells. With an initial set of cathepsin L inhibitors, we demonstrated (i) potent and selective cathepsin L inhibitory activity versus recombinant enzyme and (ii) cell-contained enzyme; (iii) lack of cytotoxicity; and (iv) inhibition of pseudotype Ebola virus entry into 293T cells. These results validate this host enzyme at the cellular level as a target for anti-Ebola therapy for the first time. In Phase I, we will design and build new potent and selective inhibitors of cathepsin L by rational drug design and structure-based drug design methods, employing a rapid sensitive fluorescence assay for cathepsin L inhibition and inhibitor-enzyme co-crystal structures. We will prioritize the most potent inhibitors for their selectivity (eg, minimal affinity for cathepsin B and calpain), minimal cytotoxicity, cell permeability, favorable ADME properties and efficacy in an Ebola pseudotype assay as well as a virulent wild-type Ebola virus tested in a BSL4 facility. We will establish Proof of Concept by demonstrating the activity of pseudotype virus inhibitors in an infectious Ebola virus plaque assay in a BSL4 containment facility at USMARIID. In Phase II, these leads will be developed into preclinical candidates and in vivo IND enabling efficacy and pharmacokinetic and toxicology studies will be performed. [unreadable] [unreadable] Ebola virus infections constitute one of the most severe threats of infection to mankind. The natural reservoir for the Ebola virus is unknown, which increases the need for therapeutic intervention. This proposal describes a rational approach for the preparation of small molecule inhibitors of Ebola virus infection. [unreadable] [unreadable] [unreadable] [unreadable]